Streptococcal ABC transporter protein

ABSTRACT

A polypeptide suitable for use in vaccination against Streptococcal infections comprises: a) the amino acid sequence of SEQ ID NO 1; b) a variant of (a) which is capable of binding an anti-MtsA antibody; or (c) a fragment of (a) or (b) of at least 6 amino acids in length which is capable of binding an anti-MtsA antibody. A vaccine composition comprises: a) the amino acid sequence of SEQ ID NO 1; b) a variant of (a) which is capable of generating an immune response to a  Streptococcus;  or (c) a fragment of (a) or (b) of at least 6 amino acids in length which is capable of generating an immune response against a  Streptococcus.

FIELD OF THE INVENTION

This invention relates to a novel protein which forms part of an ABCtransporter of S.pyogenes. The invention also relates to a streptococcusvaccine composition comprising this protein or fragments thereof.

BACKGROUND OF THE INVENTION

Streptococcus pyogenes, group A Streptococcus, is a common humanpathogen which causes a variety of diseases such as pharingitis,impetigo, scarlatina and erysipelas. More severe infections caused bythis organism are necrotizing fascitis and streptococcal toxic shocklike syndrome.

The superfamily of ABC (ATP-binding cassette) transporters comprise manydifferent systems in procaryotes and eukaryotes. This diverse group oftransporters serve many roles including transport of nutrients,translocation of signal molecules and chemotaxis. The general principleof ABC transport includes transportation of a ligand through twointegral membrane domains forming a pore, with accompanying ATPhydrolysis by two nucleotide-binding domains associated with thecytoplasmic side of the pore.

In bacteria, the translocation of ligands is preceded by interactionwith an accessory component, the periplasmic binding protein. Thisprotein binds the ligand with higher affinity, and then interacts withthe integral membrane components by releasing the ligand and allowingsubsequent transport. In gram-positive bacteria, the binding proteinhomologue is a lipoprotein attached to the cell membrane by aNH₂-terminal lipid moiety. Little is known about the ABC transporters ingram-positive bacteria. In particular, the interaction between the lipidprotein component and the integral membrane component is unclear.

A number of recent studies have looked at an ABC transporter family inStreptococcus species. Examples are Correia et al Infect. Immun (1996)64(6) 2114-2121; Fenno et al Mol.Microbiol. (1995) 15(5) 849-863; Loweet al Infect. Immun (1995) 63(2) 703-706; Kolenbrander et al Infect.Immun (1994) 62(10) 4469-4480 and Sampson et al Infect. Immun (1994)62(1) 319-324.

The genes encoding the ABC transporter form an operon consisting ofthree genes. The putative proteins encoded by these genes are ahydrophobic membrane protein, a nucleotide binding protein and a lipidprotein. The operon has been sequenced in a number of important diseasecausing organisms such as S.pneumoniae, Enterococcus faecalis, S.sanguisand S.parasanguis as well as in commensal bacteria such as S.gordoniiand Streptococcus crista. As well as playing a role in transport, theABC transporter has also been related to bacterial virulence and maymediate bacterial coaggregation, adhesion to host cells, saliva pelliclecomponents and fibrin clots.

SUMMARY OF THE INVENTION

The applicants have now identified a new protein of S.pyogenescomprising the lipoprotein of an ABC transporter. This protein is hereinreferred to as the SmtA protein or MtsA. The S.pyogenes operon isatypically organised and the polycistronic transcription is attenuated,in contrast to the previously described systems. The lipoprotein can besolubilized from the bacterial surface by proteolytic cleavage whichindicates the presence of a flexible hinge region between theNH₂-terminal lipid moiety and a more compact globular fold. Thislipoprotein and fragments thereof can be used in streptococcal vaccinecompositions and in particular against S.pyogenes.

In a first aspect, the invention provides a polypeptide which comprises:

-   -   (a) the amino acid sequence of SEQ ID NO 1,    -   (b) a variant of (a) which is capable of binding an anti-MtsA        antibody, or    -   (c) a fragment of (a) or (b) of at least 6 amino acids in length        which is capable of binding to an anti-MtsA antibody.

In another aspect, the invention relates to a vaccine compositioncomprising a polypeptide which comprises:

-   -   (a) the amino acid sequence of SEQ ID NO 1,    -   (b) a variant of (a) which is capable of generating an immune        response to a Streptococcus, or    -   (c) a fragment of (a) or (b) of at least 6 amino acids in length        which is capable of generating an immune response against a        Streptococcus.

In a further aspect, the invention relates to novel polynucleotideshaving a sequence which is:

-   -   (i) the nucleotide coding sequence of SEQ ID NO 1 or a sequence        complementary thereto,    -   (ii) a nucleotide sequence which selectively hybridises to a        said sequence (i) or fragment thereof, or    -   (iii) a nucleotide sequence which codes for a polypeptide having        the same amino acid sequence as that encoded by a said sequence        of (i) or (ii).

Polynucleotides are therefore provided which encode a polypeptide of theinvention. The invention also provides:

-   -   a recombinant vector comprising a polynucleotide of the        invention, such as an expression vector in which the        polynucleotide is operably linked to a regulatory sequence;    -   a host cell which is transformed with a polynucleotide of the        invention;    -   a process of producing a polypeptide of the invention comprising        maintaining a host cell transformed with a polynucleotide of the        invention under conditions to provide expression of the        polypeptide;    -   an antibody, monoclonal or polyclonal, specific for a        polypeptide as defined in claim 1; and    -   a method of vaccinating a patient against a Streptococcal        infection, which method comprises administering to the patient        an effective amount of a polypeptide according to the invention.

DESCRIPTION OF THE FIGURES

FIG. 1. Organization of the S.pyogenes lraI operon. The three genes inthe S.pyogenes lraI operon are, from the left (5′), mtsA (lipoprotein),mtsB (ATP-binding protein), mtsC (integral membrane protein). Arrowsindicate the location of primers used to verify the operon organization,and to create probes for each of the three genes. In the 66 bpnon-coding region between mtsA and mtsB a putative stem-loop structureis present.

FIG. 2. Proton-induced X-ray emission analysis (PIXE). PIXE analysis ofGST, GST:MtsA, MtsA and (iron-saturated) transferrin in millipore water.Results are based on two different experiments and shown in mol elementper mol protein.

FIG. 3. Binding of ⁶⁵Zn to MtsA. A. MtsA and GST were separatelyincubated with ⁶⁵Zn. The mixtures were subjected to gel filtration on aPD-10 column and 500 μl fractions were collected. Fractions were assayedfor protein content and radioactivity. Co-migration of ⁶⁵Zn and proteinwas seen in the MtsA sample (top graph) but not in the GST samples(bottom graph).

FIG. 4. Plots the results of sheep anti-MtsA antisera ELISA againstplates coated with MtsA peptides.

FIG. 5. Plots the results of sheep anti-MtsA peptide antisera ELISAagainst plates coated with MtsA protein.

FIG. 6. Plots the results of MtsA peptide blocking of sheep anti-MtsApeptide antisera ELISA against plates coated with MtsA protein.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO 1 sets out the amino acid sequence for the full length MtsApolypeptide of S.pyogenes and the nucleotide sequence encoding thisprotein. The structure of MtsA is discussed in more detail below.

SEQ ID NO 2 sets out the amino acid sequence for full length MtsA ofS.pyogenes.

SEQ ID NO 3 and SEQ ID No 4 are examples of primers which may be used inthe cloning of polynucleotides encoding MtsA.

SEQ ID NOs 5 to 9 are examples of MtsA peptides which may be used togenerate anti-MtsA antisera.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a polypeptide consisting essentially of (a) theamino acid sequence of SEQ ID NO 1; (b) a variant of the amino acidsequence of SEQ ID NO 1; or (c) a fragment of at least 6 amino acids inlength of (a) or (b). Typically the polypeptide is capable of binding ananti-MtsA antibody.

Antibody to MtsA can be raised against purified MtsA protein usingprotein purified directly from S.pyogenes expressing this protein asdescribed in more detail below. Alternatively, protein can be generatedrecombinantly. Following purification of the protein, antibody can beraised in an animal such as a rabbit and purified to generate thedesired antibody. The antibody can be monoclonal or polyclonal antibody.Preferably, the polypeptide of the invention is incorporated in avaccine composition for immunisation against a Streptococcal infection.Preferably, the antibody is neutralising antibody. Preferably, apolypeptide of the invention generates anti-MrsA antibody whenadministered in vivo and provides protection against subsequentStreptococcal infection.

Preferably, a polypeptide of the invention provides protection againstgroup A Streptococcus but may also be used to provide vaccines againstother Streptococcus such as S.pnuemoniae.

Polypeptides for incorporation into the vaccine compositions inaccordance with the invention can be identified by determining whetherthey bind to an antibody specific for MtsA. Alternatively, antibody to acandidate polypeptide can be generated by standard techniques, forexample by injection of the polypeptide into an appropriate animal andsubsequent collection and purification of antisera from such animals.Antibody which binds MtsA can then be identified by standard andcompetitive immunoassays. The antibody thus identified can then beinjected into mice to determine if it protects against a lethalchallenge with a Streptococcal strain. Alternatively, an otherwiselethal dose of a strain of Streptococcus is given in an animal modelsystem in which the animals have been given the relevant polypeptide.

As noted above a variant polypeptide (b) is one which will bind with ananti-MtsA antibody. Alternatively, a variant for incorporation into avaccine composition is one which can be used to generate an immuneresponse to provide protection against a Streptococcal infection. Avariant of SEQ ID No 2 may be a naturally occurring variant which isexpressed by another strain of S.pyogenes. Such variants may beidentified by looking for a metal transporter protein in these strainswhich has a sequence which is highly conserved compared to SEQ ID No 2.Such proteins may be identified by analysis of the polynucleotideencoding such a protein isolated from alternative strains of S.pyogenesfor example by carrying out the polymerase chain reaction using primersderived from portions of SEQ ID No 1. The Examples below demonstrateidentification of MtsA derived from a number of S.pyogenes strains.Primers hybridizing to portions of the DNA sequence of SEQ ID No 1 suchas those of SEQ ID No 3 or 4 can be used in the cloning and sequencingof MtsA from other S.pyogenes strains. MtsA antisera generated, forexample, using the peptide of SEQ ID No 8 may be used to identify MtsAexpressed by other S.pyogenes strains.

Variants of SEQ ID No 1 include sequences which vary from SEQ ID No 1but are not necessarily naturally occurring MtsA. Over the entire lengthof the amino acid sequence of SEQ ID NO 1, a variant will preferably beat least 80% homologous to that sequence based on amino acid identity.More preferably, the polypeptide is at least 85% or 90% and morepreferably at least 95%, 97% or 99% homologous to the amino acidsequence of SEQ ID NO 1 over the entire sequence. There may be at least80%, for example at least 85%, 90% or 95%, amino acid identity over astretch of 40 or more, for example 60, 100 or 120 or more, contiguousamino acids (“hard homology”).

Amino acid substitutions may be made to the amino acid sequence of SEQID NO 1, for example from 1, 2 or 3 to 10, 20 or 30 substitutions.Conservative substitutions may be made, for example, according to thefollowing table. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other: ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M NQ Polar - charged D E K R AROMATIC H F W Y

One or more amino acid residues of the amino acid sequence of SEQ ID NO1 may alternatively or additionally be deleted. From 1, 2 or 3 to 10, 20or 30 residues may be deleted, or more. Polypeptides of the inventionalso include fragments (c) of the above-mentioned sequences. Suchfragments retain the ability to bind anti-MtsA antibody. Fragments maybe at least from 10, 12, 13 or 20 to 60, 100 or 200 amino acids inlength. Particularly preferred fragments comprise;

-   -   the sequence from amino acid residue number 136 through to        residue 152 of SEQ ID NO 1, having the sequence        KQLIAKDPKNKETYEKN (SEQ ID No 5);    -   the sequence commencing at position 204 through to 22 of SEQ ID        NO 1, having the sequence EINTEEEGTPDQISSLIEK (SEQ ID No 6);    -   the sequence commencing at position 234 of SEQ ID NO 1 through        to position 249 having the sequence ESSVDRRPMETVSKDS (SEQ ID No        7);    -   the sequence commencing at position 259 of SEQ ID NO 1 through        to position 279, having the sequence TDSIAKKGKPGDSYYAMMKWN (SEQ        ID No 8); and    -   variants of these sequences.

One or more amino acids may be alternatively or additionally added tothe polypeptides described above. An extension may be provided at theN-terminus or C-terminus of the amino acid sequence of SEQ ID NO 1 orpolypeptide variant or fragment thereof. The or each extension may bequite short, for example from 1 to 10 amino acids in length.Alternatively, the extension may be longer. A carrier protein may befused to an amino acid sequence according to the invention. A fusionprotein incorporating the polypeptides described above can thus beprovided.

Polypeptides of the invention may be in a substantially isolated form.It will be understood that the polypeptide may be mixed with carriers ordiluents which will not interfere with the intended purpose of thepolypeptide and still be regarded as substantially isolated. Apolypeptide of the invention may also be in a substantially purifiedform, in which case it will generally comprise the polypeptide in apreparation in which more than 90%, e.g. 95%, 98% or 99%, by weight ofthe polypeptide in the preparation is a polypeptide of the invention.

Polypeptides of the invention may be modified for example by theaddition of histidine residues to assist their identification orpurification or by the addition of a signal sequence to promote theirsecretion from a cell where the polypeptide does not naturally containsuch a sequence. It may be desirable to provide the polypeptides in aform suitable for attachment to a solid support. For example thepolypeptides of the invention may be modified by the addition of acysteine residue.

A polypeptide of the invention above may be labelled with a revealinglabel. The revealing label may be any suitable label which allows thepolypeptide to be detected. Suitable labels include radioisotopes, e.g.¹²⁵I, ³⁵S, enzymes, antibodies, polynucleotides and linkers such asbiotin. Labelled polypeptides of the invention may be used in diagnosticprocedures such as immunoassays in order to determine the amount of apolypeptide of the invention in a sample.

Polypeptides or labelled polypeptides of the invention may be used in isserological or cell mediated immune assays for the detection of immunereactivity to said polypeptides in animals and humans. Standardprotocols can be used. The labelled polypeptide may be used to identifyand/or isolate “accessory” proteins which are involved in bindingbetween cell receptors and MtsA, by detecting the interaction of MtsA tosuch proteins.

A polypeptide or labelled polypeptide of the invention or fragmentthereof may also be fixed to a solid phase, for example the surface ofan immunoassay well or dipstick.

Such labelled and/or immobilized polypeptides may be packaged into kitsin a suitable container optionally including additional suitablereagents, controls or instructions and the like. The kits may be used toidentify MtsA inhibitors or activators.

Such polypeptides and kits may also be used in methods of detection ofantibodies to the MtsA protein by immunoassay.

Immunoassay methods are well known in the art and will generallycomprise:

-   -   (a) providing a polypeptide comprising an epitope bindable by an        antibody against said protein;    -   (b) incubating a biological sample with said polypeptide under        conditions which allow for the formation of an antibody-antigen        complex; and    -   (c) determining whether antibody-antigen complex comprising said        polypeptide is formed.

The proteins of the present invention may be isolated from S.pyogenesexpressing the protein. Proteins and peptides of the invention may beprepared as fragments of such isolated proteins. The proteins andpeptides of the invention may also be made synthetically or byrecombinant means. The amino acid sequence of proteins and polypeptidesof the invention may be modified to include non-naturally occurringamino acids or to increase the stability of the compound. When theproteins or peptides are produced by synthetic means, such amino acidsmay be introduced during production. The proteins or peptides may alsobe modified following either synthetic or recombinant production.

The proteins or peptides of the invention may also be produced usingD-amino acids. In such cases the amino acids will be linked in reversesequence in the C to N orientation. This is conventional in the art forproducing such proteins or peptides.

A number of side chain modifications are known in the art and may bemade to the side chains of the proteins or peptides of the presentinvention. Such modifications include, for example, modifications ofamino acids by reductive alkylation by reaction with an aldehydefollowed by reduction with NaBH₄, amidination with methylacetimidate oracylation with acetic anhydride.

The polypeptides of the invention may be introduced into a cell by insitu expression of the polypeptide from a recombinant expression vector.The expression vector optionally carries an inducible promoter tocontrol the expression of the polypeptide.

Such cell culture systems in which polypeptides of the invention areexpressed may be used in assay systems.

A polypeptide of the invention can be produced in large scale followingpurification by high pressure liquid chromatography (HPLC) or othertechniques after recombinant expression as described below.

A polynucleotide of the invention typically is a contiguous sequence ofnucleotides which is capable of hybridising selectively with the codingsequence of SEQ ID NO 1 (nucleotides 1 to 861) or to the sequencecomplementary to that coding sequence. Polynucleotides of the inventioninclude variants of the coding sequence of SEQ ID NO 1 which encode theamino acid sequence of SEQ ID NO 1 and variants and fragments of thatsequence which are recognized by antibody to MtsA.

A polynucleotide of the invention and the coding sequence of SEQ ID NO 1can hybridize at a level significantly above background. Backgroundhybridization may occur, for example, because of other cDNAs present ina cDNA library. The signal level generated by the interaction between apolynucleotide of the invention and the coding sequence of SEQ ID NO 1is typically at least 10 fold, preferably at least 100 fold, as intenseas interactions between other polynucleotides and the coding sequence ofSEQ ID NO 1. The intensity of interaction may be measured, for example,by radiolabelling the probe, e.g. with ³²P. Selective hybridization istypically achieved using conditions of medium to high stringency (forexample 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C. to about 60° C.).

A nucleotide sequence capable of selectively hybridizing to the DNAcoding sequence of SEQ ID NO 1 or to the sequence complementary to thatcoding sequence will be generally at least 80%, preferably at least 90%and more preferably at least 95%, homologous to the coding sequence ofSEQ ID NO 1 or its complement over a region of at least 20, preferablyat least 30, for instance at least 40, 60 or 100 or more contiguousnucleotides or, indeed, over the full length of the coding sequence.Thus there may be at least 85%, at least 90% or at least 95% nucleotideidentity over such regions.

Any combination of the above mentioned degrees of homology and minimumsize may be used to define polynucleotides of the invention, with themore stringent combinations (i.e. higher homology over longer lengths)being preferred. Thus for example a polynucleotide which is at least 85%homologous over 25, preferably over 30, nucleotides forms one aspect ofthe invention, as does a polynucleotide which is at least 90% homologousover 40 nucleotides.

For example the UWGCG Package provides the BESTFIT program which can beused to calculate homology (for example used on its default settings).

(Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUPand BLAST algorithms can be used to calculate homology or line upsequences (such as identifying equivalent or corresponding sequences(typically on their default settings), for example as described inAltschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990)J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSP's containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen: the cumulative alignment score falls off by the quantity X fromits maximum achieved value; the cumulative score goes to zero or below,due to the accumulation of one or more negative-scoring residuealignments; or the end of either sequence is reached. The BLASTalgorithm parameters W, T and X determine the sensitivity and speed ofthe alignment. The BLAST program uses as defaults a word length (W) of11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc.Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation(E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl.Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by theBLAST algorithm is the smallest sum probability (P(N)), which providesan indication of the probability by which a match between two nucleotideor amino acid sequences would occur by chance. For example, a sequenceis considered similar to another sequence if the smallest sumprobability in comparison of the first sequence to the second sequenceis less than about 1, preferably less than about 0.1, more preferablyless than about 0.01, and most preferably less than about 0.001.

Polynucleotides of the invention may comprise DNA or RNA. They may alsobe polynucleotides which include within them synthetic or modifiednucleotides. A number of different types of modification topolynucleotides are known in the art. These include methylphosphate andphosphorothioate backbones, addition of acridine or polylysine chains atthe 3′ and/or 5′ ends of the molecule. For the purposes of the presentinvention, it is to be understood that the polynucleotides describedherein may be modified by any method available in the art.

Polynucleotides of the invention may be used to produce a primer, e.g aPCR primer, a primer for an alternative amplification reaction, a probee.g. labelled with a revealing label by conventional means usingradioactive or non-radioactive labels, or the polynucleotides may becloned into vectors. Such primers, probes and other fragments will be atleast 15, preferably at least 20, for example at least 25, 30 or 40nucleotides in length, and are also encompassed by the termpolynucleotides of the invention as used herein. SEQ ID Nos 3 and 4 areexamples of primers of the invention.

Polynucleotides such as a DNA polynucleotide and primers according tothe invention may be produced recombinantly, synthetically, or by anymeans available to those of skill in the art. They may also be cloned bystandard techniques. The polynucleotides are typically provided inisolated and/or purified form,

In general, primers will be produced by synthetic means, involving astep wise manufacture of the desired nucleic acid sequence onenucleotide at a time. Techniques for accomplishing this using automatedtechniques are readily available in the art.

Longer polynucleotides will generally be produced using recombinantmeans, for example using PCR (polymerase chain reaction) cloningtechniques. This will involve making a pair of primers (e.g. of about15-30 nucleotides) to a region of the mtsA gene which it is desired toclone, bringing the primers into contact with DNA obtained from abacterial cell, performing a polymerase chain reaction under conditionswhich bring about amplification of the desired region, isolating theamplified fragment (e.g. by purifying the reaction mixture on an agarosegel) and recovering the amplified DNA. The primers may be designed tocontain suitable restriction enzyme recognition sites so that theamplified DNA can be cloned into a suitable cloning vector.

Such techniques may be used to obtain all or part of the mtsA genesequence described herein. Genomic clones containing the mtsA gene andits promoter region may also be obtained in an analogous manner,starting with genomic DNA from a bacterial cell.

Although in general the techniques mentioned herein are well known inthe art, reference may be made in particular to Sambrook et al, 1989.

Polynucleotides or primers of the invention may carry a revealing label.

Suitable labels include radioisotopes such as ³²P or ³⁵S, enzyme labels,or other protein labels such as biotin. Such labels may be added topolynucleotides or primers of the invention and may be detected usingtechniques known per se.

Polynucleotides or primers of the invention or fragments thereof,labelled or unlabelled, may be used by a person skilled in the art innucleic acid-based tests for detecting or sequencing MtsA in a sample.

Such tests for detecting generally comprise bringing a sample containingDNA or RNA into contact with a probe comprising a polynucleotide orprimer of the invention under hybridizing conditions and detecting anyduplex formed between the probe and nucleic acid in the sample. Suchdetection may be achieved using techniques such as PCR or byimmobilizing the probe on a solid support, removing nucleic acid in thesample which is not hybridized to the probe, and then detecting nucleicacid which has hybridized to the probe. Alternatively, the samplenucleic acid may be immobilized on a solid support, and the amount ofprobe bound to such a support can be detected.

The probes of the invention may conveniently be packaged in the form ofa test kit in a suitable container. In such kits the probe may be boundto a solid support where the assay formats for which the kit is designedrequires such binding. The kit may also contain suitable reagents fortreating the sample to be probed, hybridizing the probe to nucleic acidin the sample, control reagents, instructions, and the like.

Polynucleotides of the invention can be incorporated into a recombinantreplicable vector. The vector may be used to replicate the nucleic acidin a compatible host cell. Thus in a further embodiment, the inventionprovides a method of making polynucleotides of the invention byintroducing a polynucleotide of the invention into a replicable vector,introducing the vector into a compatible host cell, and growing the hostcell under conditions which bring about replication of the vector. Thevector may be recovered from the host cell. Suitable host cells aredescribed below in connection with expression vectors.

Preferably, a polynucleotide of the invention in a vector is operablylinked to a control sequence which is capable of providing for theexpression of the coding sequence by the host cell, i.e. the vector isan expression vector. Such expression vectors can be used to express thepolypeptide of the invention.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is ligated in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences.

Such vectors may be transformed into a suitable host cell to provide forexpression of a polypeptide of the invention. Thus, a polypeptideaccording to the invention can be obtained by cultivating a host celltransformed or transfected with an expression vector as described aboveunder conditions to provide for expression of the polypeptide, andrecovering the expressed polypeptide.

The vectors may be for example, plasmid, virus or phage vectors providedwith an origin of replication, optionally a promoter for the expressionof the said polynucleotide and optionally a regulator of the promoter.The vectors may contain one or more selectable marker genes, for examplean ampicillin resistance gene in the case of a bacterial plasmid.Promoters and other expression regulation signals may be selected to becompatible with the host cell for which the expression vector isdesigned.

Host cells transformed (or transfected) with the polynucleotides orvectors for the replication and expression of polynucleotides of theinvention will be chosen to be compatible with the said vector andpreferably will be bacterial such as E. coli. Alternatively they may becells of a human or animal cell line such as CHO or COS cells, or yeastor insect cells. The cells may also be cells of a non-human animal suchas a sheep or rabbit or plant cells.

The polypeptides of the invention are useful for vaccinating against aStreptococcal infection, for example-against group A Streptococcus orS.pneumoniae. A vaccine of the invention comprises a suitablepolypeptide and a pharmaceutically acceptable carrier or diluent. Thepreparation of vaccines which contain an immunogenic polypeptide(s) asactive ingredient(s), is known to one skilled in the art. Typically,such vaccines are prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified, or the protein encapsulated in liposomes. The activeimmunogenic ingredients are often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol, or the like and combinations thereof. In addition, if desired,the vaccine may contain minor amounts of auxiliary substances such aswetting or emulsifying agents, pH buffering agents, and/or adjuvantswhich enhance the effectiveness of the vaccine.

Examples of adjuvants which may be effective include but are not limitedto: aluminium hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamin(thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637,referred to as nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutamnyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against an immunogenicpolypeptide containing MtsA antigenic sequence resulting fromadministration of this polypeptide in vaccines which are also comprisedof the various adjuvants.

The vaccines are conventionally administered parentally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkylene glycols or triglycerides; such suppositories maybe formed from mixtures containing the active ingredient in the range of0.5% to 10%, preferably 1% to 2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccarine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10% to 95% of activeingredient, preferably 25% to 70%. Where the vaccine composition islyophilised, the lyophilised material may be reconstituted prior toadministration, e.g. as a suspension. Reconstitution is preferablyeffected in buffer.

Capsules, tablets and pills for oral administration to a patient may beprovided with an enteric coating comprising, for example, Eudragit “S”,Eudragit “L”, cellulose acetate cellulose acetate phthalate orhydroxypropylmethyl cellulose.

The polypeptides of the invention may be formulated into the vaccine asneutral or salt forms. Pharmaceutically acceptable salts include theacid addition salts (formed with free amino groups of the peptide) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids such as acetic, oxalic,tartaric and maleic. Salts formed with the free carboxyl groups may alsobe derived from inorganic bases such as, for example, sodium, potassium,ammonium, calcium, or ferric hydroxides, and such organic bases asisopropylamine, trimethylamine, 2-ethylamino ethanol, histidine andprocaine.

The vaccines are administrated in a manner compatible with the dosageformulation and in such amount as will be prophylactically effective.The quantity to be administered, which is generally in the range of 5 μgto 100 mg, preferably 250 μg to 10 mg, of polypeptide (antigen) perdose, depends on a number of factors. These include the subject to betreated capacity of the subject's immune system to synthesizeantibodies, and the degree of protection desired. Precise amounts ofactive ingredient required to be administered may depend on thejudgement of the practitioner and may be peculiar to each subject.

The vaccine may be given in a singe dose schedule, or preferably in amultiple dose schedule. A multiple dose schedule is one in which aprimary course of vaccination may be with 1-10 separate doses, followedby other doses given at subsequent time intervals required to maintainand or reinforce the immune response, for example at 1 to 4 months for asecond dose, and if needed, a subsequent dose(s) after several months.The dosage regimen will also, at least in part, be determined by theneed of the individual and be dependent upon the judgement of thepractitioner.

The nucleotide sequences of the invention and expression vectors canalso be used as vaccine formulations as outlined above. The vaccines maycomprise naked nucleotide sequences or be in combination with cationiclipids, polymers or targeting systems. The vaccines may be delivered byany technique suitable for delivery of nucleic acid vaccines.

The immunogenic polypeptides prepared as described above can be used toproduce antibodies, both polyclonal and monoclonal. If polyclonalantibodies are desired, a selected mammal (e.g., mouse, rabbit, goat,horse, etc.) is immunised with an immunogenic polypeptide of theinvention. Serum from the immunised animal is collected and treatedaccording to known procedures. If serum containing polyclonal antibodiesto the polypeptide contains antibodies to other antigens, the polyclonalantibodies can be purified by immunoaffinity chromatography. Techniquesfor producing and processing polyclonal antisera are known in the art.

Monoclonal antibodies directed against Streptococcal epitopes in thepolypeptides of the invention can also be readily produced by oneskilled in the art. The general methodology for making monoclonalantibodies by hybridomas is well known. Immortal antibody-producing celllines can be created by cell fusion, and also by other techniques suchas direct transformation of B lymphocytes with oncogenic DNA, ortransfection with Epstein-Barr virus. Panels of monoclonal antibodiesproduced against polypeptides of the invention can be screened forvarious properties; i.e., for isotype and epitope affinity. Preferablythe antibody is specific for a MtsA protein epitope.

Antibodies, both monoclonal and polyclonal, which are directed againstpolypeptides of the invention are particularly useful in diagnosis, andthose which are neutralising are useful in passive immunotherapy.Monoclonal antibodies, in particular, may be used to raise anti-idiotypeantibodies. Anti-idiotype antibodies are immunoglobulins which carry an“internal image” of the antigen of the infectious agent against whichprotection is desired.

Techniques for raising anti-idiotype antibodies are known in the art.These anti-idiotype antibodies may also be useful for treatment ofStreptococci, as well as for an elucidation of the immunogenic regionsof polypeptides of the invention.

It is also possible to use fragments of the antibodies described above,for example, Fab fragments. Antibodies generated to a peptide of theinvention may be administered to an individual to treat GAS infection bypassive immuno therapy. The antibodies of the invention may beformulated with a pharmaceutically acceptable carrier and delivered inthe same way as set out above for the vaccine compositions. Preferablythe antibody is administered in an amount effective to ameliorate GASinfection in the individual.

The following Examples illustrate the invention.

EXAMPLES

Experimental Procedures Used in the Following Examples

Bacterial Cultures

The S.pyogenes strains used in this study (serotypes M1, M4, M9, M12,M49) are from the World Health Organization Centre for references andresearch on Streptococci, Institute of Hygiene and Epidemiology, Prague,Czech Republic. The S.pyogenes strain SF370 is being sequenced in theStreptococcal Genome Project, and can be obtained from ATCC (700294).Streptococci were grown in Todd-Hewitt broth (Difco, Detroit, Mich.),supplemented with 0.2% yeast extract (Difco) in 5% CO₂ at 37° C. E.colistrain BL21 (Pharmacia Biotech, Uppsala, Sweden) was grown inLuria-Bertani or 2X YT broth or agar, aerobically at 37° C. andsupplemented with 100 μg/ml ampicillin (Sigma, St Louis, Mo.) or glucosewhere appropriate.

PCR, Cloning Procedures, and Sequencing

Chromosomal DNA from S.pyogenes strains was extracted as described inPitcher et al, Lett. App. Microbiol 1989 8 p151-156 modified in theinitial incubation step by addition of 1000 U/ml of mutanolysin (Sigma)and 100 mg/ml lysozyme (Sigma). Oligonucleotide primers were designed byusing sequence information from the Streptococcal Genome Projectdatabase, together with published sequences from other Streptococcusspp. Primers 5′-TAG-TAG-CGA-ATT-CGT-CGA-CTG-GCG-CTA-3′ and5′-AGC-ACA-ACT-CGA-GAA-TCG-CTG-TGC-TTT-A-3′ enclose almost the whole ofmtsA (excluding the signal peptide and the NH₂-terminal cysteineresidue) and were designed with an EcoRI and XhoI restriction site,respectively. Primers5′-GAT-TAC-AAC-TAA-CAA-TCT-TTG-TGT-GAC-C-3′,5′-TTG-ACA-AGG-TAT-CAA-CAG-TAA-ATA-CCT-C-3′,5′-ATG-TCA/T-CTC/T-ATG-GGA/G/T-GAT-GCC-ATC-3′,and 5′-TTA/G-GCA-TAT/G-AG/AA-TAA/G-GCC/T-GTC-GC-3′ were designed frominternal segments of the genes mtsB and mtsC. PCR experiments wereperformed using Taq polymerase (Gibco-BRL, Gaithersburg, Md.), exceptfor cloning purposes, when TaqPlus Precision™ (Strategene, La Jolla,Calif.) was used. The PCR product corresponding to mtsA was gel-purifiedprior to cloning, using Sephaglas™ Bandprep Kit (Pharmacia Biotech). ThePCR profile consisted of 30 cycles at 94° C. for 1 min, 50° C. for 1min, and 72° C. for 2 min, followed by a final extension at 72° C. for 7min. Plasmid purification, restriction enzyme digestions, ligation,electroporation and screening of transformants were all performedaccording to standard procedures, or when applicable, according toinstructions in the GST fusion protein kit (Pharmacia). Sequencing ofthe cloned insert was performed on an ABI-470 Prism with dyed dideoxyterminators, at Innovagen AB, Lund, Sweden.

Overexpression and Purification of Recombinant MtsA

An E.coli strain carrying the recombinant plasmid pGEX-5X-3:mtsA wasgrown at 37° C. overnight. This preculture was then inoculated 1:100 inprewarmed 2×YT containing 100 μg/ml ampicillin. The culture was grown at30° C. until OD₆₀₀=1.2. Induction was then started by adding 0.5 mM IPTG(Promega, Madison, Wis.). After an additional 4 h of incubation thebacteria were pelleted by centrifugation at 8000×g for 10 min, washed,and resuspended in PBS buffer, and lysed by sonication with a Bransonsonifier B15 (Heimstatt, Germany). Triton X-100 was added to a finalconcentration of 1%, and the sample was gently mixed for 30 min. Celldebris was pelleted by centrifugation at 12000×g for 10 min, and thesupernatant was applied to a pre-equilibrated glutathione-sepharosesuspension. All washes were performed in batch format, by centrifugationat 1000×g for 5 min. GST:MtsA fusion protein was eluted with reducedglutathione. Alternatively, factor Xa cleavage (Pharmacia Biotech) wasperformed in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl₂, overnight(1 U factor Xa/ml bacterial sonicate supernatant). Cleaved MtsA proteinwas then eluted in repeated steps with PBS.

RNA Methods

Total RNA from S.pyogenes was purified using Fastprep™ cell disrupter(Savant, Holbrook, N.Y.). Briefly, bacteria were cultured in THY mediumuntil mid-logarithmic or early stationary phase, harvested bycentrifugation at 3,800×g for 10 min at 4° C., and resuspended in PBS,followed by disruption for 2×30 seconds at setting 6.0 using FastRNA™kit with glass beads (BIO 101, Vista, Calif.) according to themanufacturers instructions.

For Northern blot experiments, RNA was separated on 1% agarose in1×HEPES buffer, blotted onto Hybond-N filters (Amersham, Amersham, UK),and hybridized with 600-900 bp long DNA probes specific for mtsA, mtsBand mtsC (see above). The PCR products (see above) were purified on aMicroSpin™ S-200 HR column (Pharmacia, Uppsala, Sweden), andradiolabelled with [α-³²P]dATP using the Megaprime™ kit (Amersham). Toverify that equal amounts of RNA were present on the filter, it was alsoprobed with a radiolabelled 800 bp probe specific for 16S rRNA.

Protein Methods

Protein samples were separated by 12% SDS-PAGE. Gels were then soaked inblotting buffer (20% ethanol, 200 mM glycine, 25 mM Tris), and proteinswere blotted to an Immunobilon-P™ PVDF-membrane (Millipore). Membraneswere then blocked in 15 ml of PBS, 0.05% Tween-20, 5% (w/v) skim milkfor 20 min at room temperature. The primary antibody (see below) wasadded, diluted 1:1000 in PBS, 0.05% Tween-20 (PBST), and the membranewas incubated at 37° C. for 30 mins. Membranes were then washed in PBST,3×5 min at 37° C. Horseradish peroxidase-conjugated antirabbit goatantibodies (Bio-Rad, Bio-Rad laboratories Calif.) diluted 1:3000 in PBSTwere added and the membrane was incubated for 30 min at 37° C., and thenwashed as above.

Proteolytic digestion of bacteria was performed on overnight cultures ofS. pyogenes. Bacteria were pelleted by centrifugation at 3000×g for 5min, at 8° C., resuspended in cleavage buffer (0.01 M Tris-HCl, pH 8),and washed twice as above before being resuspended in cleavage buffer.Papain (Sigma) was added (0-200 μg/2×10⁹ bacteria), and digestion wasinitiated by the addition of L-cysteine (55 mM). The suspension wasincubated at 37° C. for 1 h. The digestion was stopped by addingIodoacetamide to a final concentration of 12 μM. Bacteria were thenpelleted by centrifugation at 4800×g for 10 min, and the supernatant wasremoved and filtered through a 0.2 μm Acrodisc (Gelman Sciences, AnnArbor, Mich.). After freezing and thawing, samples precipitatedspontaneously. This was used as a convenient method of concentration, asSDS-PAGE analysis indicated that the precipitate contained all theproteins present in the original solubilized sample. Enzymatic digestionwith streptococcal cysteine proteinase was performed essentially asdescribed in Berge and Björck, J. Biol Chem (1995) 270 9862-9867.

Metal Assays

Samples for PIXE analysis were prepared as follows. One ml of GST, MtsAfusion, and MtsA suspended in PBS was ultrafiltrated in Centricon-10cells (Amicon, Inc., Beverly, Mass.) four times, each time reducing thevolume to less than one tenth, and adding Millipore water up to 1.5 mlafter the first three spins. The final protein concentration wasdetermined using Coomassie® Protein Assay Reagent kit (Pierce, Rockford,Ill.). Bovine iron-saturated holo-Transferrin (Sigma) was suspended inMillipore water. Then, 20 and 60 μg of GST, MtsA fusion and MtsA,respectively, in 25 μl of Millipore water was added to a Kimfoil(Kimberley Clark), mounted on a plastic frame, and allowed to dry. 200μg of transferrin in 25 μl of Millipore water was similarly prepared. Atthe Lund Nuclear Microprobe the samples were placed in the vacuumirradiation chamber and bombarded with a 2 nA proton beam, having anenergy 2.55 MeV, accumulating a beam charge of 0.6 μC. Thecharacteristic X-rays emitted were detected using a Kevex 50 mm² Si(Li)X-ray detector, while data were collected using a CAMAC/Mac-computersystem equipped with KMAX (Sparrow) software. Elemental standards (Fe,Co, Ni) were analysed in the same batch to verify quantification.

Gel filtration experiments were performed with PD-10 columns (PharmaciaBiotech). 20 μg protein (GST or MtsA) in PBS, supplemented with 0.25 MNaCl, was incubated overnight at 4° C. with approx. 0.5 μCi of ⁵⁴Mn,⁶⁵Zn or ⁵⁹Fe (Amersham). The sample was applied to a gel filtrationcolumn, preequilibrated with PBS, 0.25 M NaCl, and 0.5 ml fractions wereeluted with the same buffer.

Each fraction was then assayed for protein content, using Coomassie®Protein Assay Reagent kit (Pierce), and radioactivity, by adding ReadySafe™ scintillation fluid (Beckman Instruments, Fullerton, Calif.) andcounting β-emission on a Beckman Instruments LS6000TA.

Slot blot experiments were performed by applying 10, 1 and 0.1 μg ofprotein (MtsA fusion and GST) to a nitrocellulose membrane, using a slotblot apparatus (Millipore). Membranes were equilibrated in 0.1 M citratebuffer, pH 6.2, (appr. 0.1 M Na⁺), or 0.1 M maleic acid, pH 6.5, 0.1 MNaCl (for iron-binding) and then suspended in the same buffer with 1μCi/ml of radioisotope (⁵⁹Fe, ⁵⁴Mn or ⁶⁵Zn), for 1 h at room temperatureand with gentle mixing. In competition experiments 1 mM of Fe(II)sulfate, Fe(III) citrate. MnCl₂, CuCl₂ or ZnCl₂ was added. Then,membranes were washed for 2×15 min in buffer, and exposed on aphosphoimaging plate (Fuji Photo Film Co. Ltd., Japan). Quantificationof bound radioactivity was performed using the Bio-Imaging AnalyzerBAS2000 program package (Fuji Photo Film).

Other Methods

NH₂-terminal sequencing (Edman degradation) was performed at theBiomedical Service Unit, Lund University. The peptide QDPHEYEPLPEDV SEQID No 9 was synthesized, analysed for purity and correct sequence.

Rabbits were immunized with peptide-KLH conjugates. Primary immunizationwas performed with 100 μg peptide-KLH conjugate in Freunds completeadjuvant (Sigma). The two booster immunizations were done at weeks 4 and6, using 100 μg peptide-KLH conjugate in Freunds incomplete adjuvant(Sigma).

Example 1 Identification and Sequence Analysis of a S. pyogenes Memberof the lraI Family

Predicted amino acid sequences of the proteins encoded by the variouslraI operons were used to search (tBLASTn) the Streptococcal GenomeProject database, at the time of 95% completion. The products of fouradjacent open reading frames (ORF), showed strong homology to the LraIproteins. A frame shift split one gene (lipoprotein) in two ORF's butsubsequent sequencing (see below) of a serotype M1 strain showed thatthe database sequence was incorrect, containing a single base insertionin a region of reduced database sequence quality. The (corrected) threeORFs were named mtsABC (streptococcal metal transporter).

The three ORFs encode proteins typical of an ABC transport system. MtsAcontains a putative hydrophobic signal peptide and a consensus sequence(LXXC) typical of NH₂-terminal lipid linkage bacterial lipoproteins.MtsB has an ATP-binding cassette, while MtsC is a highly hydrophobicprotein with 6-7 potential transmembrane domains. Homologies between theputative proteins of S.pyogenes and their counterparts in otherStreptococcus spp. were in the same range as previously described forthe lraI family, as noted in the Table below. The percentages indicatesequence identity at the protein level. An “*” indicates only partialsequences available. ATP-binding Integral Bacterium Lipoprotein proteinmembrane protein S. pneumoniae 72%  51%* 70% S. gordonii 73% 56% 78% S.parasanguis 72% 54% 75% S. sanguis 74%  75%* S. crista 72%  73%* E.faecalis 55%

Notably, the arrangement of the genes in the S. pyogenes operon wasatypical. The lipoprotein gene is at the 5′-end of the operon, whereasin all other operons it is at the 3′-end. A publication on anoligopeptide permease ABC-transporter in S. pyogenes Polbielski et alMol. Microbiol. 1996 21(5) 1087-1099 showed a differential transcriptionof the lipoprotein component. A putative stem-loop structure (−60.2 kJmol⁻¹) (FIG. 1B) similar to Rho-independent transcription terminatorswas identified in the 66 bp non-coding region between MtsA and MtsBC.

The atypical organization was verified by PCR experiments. Forward andreverse primer pairs from each of the three genes were used to amplifyproducts with template DNA from five different strains (serotypes M1,M4, M9, M12, and M49) of S. pyogenes, in combinations so as to allowevery possible arrangement of the three genes in the operon. Allexperiments gave single products of a size consistent only with theatypical operon seen in the database (data not shown).

The GENBANK database was searched for homologues of MtsA, MtsB and MtsC,using the BLAST algorithm. The highest scoring matches were all ABCtransporters specific for di-or trivalent cations. Specifically, severaliron-siderophore transport systems showed a high degree of homology tothe streptococcal proteins. MtsA has a 34% identify with YfeA (Beardenet at J. Bacteriol 1998 180(5) 1135-1147), the periplasmic bindingprotein of an iron (chelated) transport system in Yersinia pestis (alsoin Haemophilus influenzae). An inversely directed search, using YfeA tosearch for homologues in the Streptococcal Genome database, identifiedMtsA as the best homologue on the streptococcal genome. TroA (Hardman etal Gene 1997 197 47-64) from Treponema pallidum also shows homology (27%id. and 17% sim.) with MtsA, and is part of an ABC transporter operonflanked by iron-regulated transcription factors. Additionally, arecently described iron-regulated ABC transporter in Staphylococcusepidermidis (Cockayne et al Infect Immun 1998 66 3767-3774) has a highdegree of homology (the S. epidermidis lipoprotein is 52% identical toMtsA) to lraI protein, and should possibly be included in this family.

Two homologous ABC transporters not involving iron were also found: themanganese transporter MntABC (Bartsevich and Pakrasi EMBO J 1995 14(9)1845-1853) from Synechococcus cystis, and the zinc transporter AdcCBAfrom S. pneumoniae, where MtsA showed appr. 30% identitv with thecorresponding proteins, MntC and AdcA. A comparison of MtsC withconserved motifs of integral membrane proteins from ABC transporters(Saurin et al Mol Microbiol 1994 12 993-1004) showed that the bestfitting (21% id. and 21% sim. on 43 aa) motif was from the cluster ofiron-siderophore transporters.

Example 2 Transcriptional Analysis of mtsABC

In order to investigate the transcription of the three genes in the S.pyogenes lraI operon, probes corresponding to internal sequences ofmtsA, mtsB and mtsC were produced and labelled. Total RNA was extractedfrom serotype M1 S. pyogenes bacteria grown to mid-logarithmic and earlystationary phase. Northern blot experiments (data not shown) showed thatall three probes reacted weakly with a transcript approximately 2.5 kbin size when RNA from mid-log phase bacteria was used, consistent with apolycistronic transcription of the operon. However, the mtsA probe alsoreacted with a shorter transcript, approximately 1 kb in size. Thisshorter transcript was present in higher (10 -20 times) amounts than thepolycistronic transcript, suggesting that the stem-loop structure in thenon-coding region between mtsA and mtsB can terminate transcription. TheMtsA probe showed weak reactivity with the short transcript when RNAfrom bacteria in early stationary phase was used, whereas the two otherprobes did not hybridize at all. A control hybridization with a probefor 16S rRNA was done to verify that RNA levels in the samples (mid-logand early stationary phase of growth) were equal (data not shown). Takentogether, these results indicate that the lipoprotein is expressed inhigher quantities than the ATPase and hydrophobic membrane protein.

Example 3 Surface Localization of the MtsA Lipoprotein

We produced the synthetic peptide QDPHEYEPLPEDV, spanning a highlyconserved region of MtsA, predicted to have good antigenicity. A rabbitwas immunized with the peptide, and following three boosters the serumshowed good reactivity with the peptide in ELISA, as compared withpreimmune serum (data not shown). Then, a proteolytic digestion of S.pyogenes bacteria was performed, to investigate whether a proteinfragment from the lipoprotein could be identified using the peptideantiserum. The protein fragments released by proteolytic digestion weresubjected to SDS-PAGE and then transferred to PVDF-membrane by Westernblotting.

Immunodetection with the peptide antiserum identified a protein fragmentwith an apparent molecular mass of 36 kDa, solubilized at highconcentrations of papain (data not shown). The protein seemed fairlyresistant to proteolytic digestion, since it remained at the sameposition even at high papain concentrations when most of the proteinshad shifted to the low molecular weight range, supposedly degraded bythe excess of protease. Protein from the 36 kDa band was subjected toNH₂-terminal amino acid sequencing. The result (KSDKLKVVAT, aa 1-10 ofthe 36 kDa papain fragment) showed a 90% identity to a region very closeto the NH₂-terminus in the predicted MtsA protein (amino acids 30-39) ofthe database (ESDKLKVVAT), and a 100% identity to the predicted MtsAprotein (amino acids 30-39) from the sequence of the strain studied (seebelow). The predicted molecular mass of the mature MtsA polypeptide was32 kDa. Thus, papain seems to cleave MtsA very close to the protein'spredicted NH₂-terminal lipid anchor, liberating almost the wholepolypeptide from the bacterial cell surface. A similar papain digestionand western blot was also performed with the strain SF370 sequenced inthe Streptococcal Genome Project, with the same result.

Bacterial growth media from overnight cultures of S. pyogenes was alsoexamined for presence of MtsA, since bacterial lipoproteins aresometimes found in medium as well. No reactivity with the peptideantiserum was seen in TCA-precipitated proteins from medium. Also, milddetergent treatment of bacteria failed to solubilize any proteinreacting with the antiserum.

S. pyogenes produces and secretes a cysteine proteinase, SCP. Thisprotease has previously been shown to release functionally activefragments of streptococcal surface proteins. We performed proteolyticdigestions of S. pyogenes with purified SCP. Solubilized proteins werevisualized by SDS-PAGE and analysed by immunoblotting. There was noreactivity with the antiserum. To exclude that MtsA had been completelydegraded, a double digestion was performed, first with SCP and then withpapain. The amount of MtsA released, as judged by staining andimmunoblotting, was unaffected by pretreatment with SCP (data notshown).

Example 4 Cloning and Purification of Recombinant MtsA

The mtsA gene from strain AP1 was PCR-amplified and cloned into pGEX andsequence analysis confirmed the presence of MtsA, showing 98% (aminoacid) identity with the database sequence, well in line with what couldbe expected from two different strains of the same serotype compare psaAfrom S. pneumoniae Berry and Paton Infect. Immun. 1996 64(12) 5255-5262.There was no frame shift in the sequenced ORF, indicating that thepreliminary database sequence indeed contained an incorrect baseinsertion (see above). Also, the supposed error is found in a regionwhere the database sequence is less accurate. MtsA was purified byaffinity chromatography of over expressed MtsA fusion and subsequentproteolytic cleavage with factor Xa. The purified MtsA had an apparentmolecular mass of 36 kDa, and comprised >95% of the protein content inthe sample (data not shown).

Computer predictions (Robson-Garnier and Chou-Fasman algorithms) of MtsAsecondary structure suggest a predominantly α-helical structure (45-60%of the protein). CD-spectroscopy of MtsA confirmed these predictions(data not shown).

Example 5 Analysis of Trace Element Content and Metal Binding Propertiesof MtsA

A recent publication on a periplasmic molybdate-binding protein of E.coli Rech et al J. Biol Chem 1996 271 2557-2562 showed that binding ofthe ligand changed the migration of the protein in native PAGE. Similarexperiments were performed with MtsA, but no consistent effects onmigration properties was seen with any of the tested potential ligands(Mn(II), Fe(III), Cu(II), Zn(II), Co(II), Ca(II)), nor with EDTA (datanot shown). We performed a quantitative analysis of trace elementcontent in MtsA, MtsA fusion, GST and (iron-saturated) transferrin bythe use of a highly specific and sensitive technique, proton-inducedX-ray emission (PIXE). PIXE analysis indicated that iron was present inapproximately 1.5 molar ratio compared to protein in the MtsA sample(FIG. 2). Little or no iron was detected in the other samples, exceptfor transferrin. Among other trace elements only copper was present insignificant amounts. Both MtsA fusion and MtsA contained copper inapproximately 0.5 molar ratio to protein. In a similar analysis, using a0.1 M Tris-Ac buffer, pH 7.5, a significant Zn content was found (datanot shown).

In addition, 30 μM solutions of GST and MtsA were analysed for ironcontent at a routine clinical chemistry laboratory. The sample with MtsAcontained 20 μM iron, indicating a 67% saturation of the protein. Torule out that iron contamination from the factor Xa solution used forcleaving the fusion protein was affecting the results, a samplecontaining cleavage solution, reduced glutathione, and PBS was analysedfor iron content. Iron concentration in that sample was <5 μM (detectionlimit).

MtsA was subjected to proteolytic digestion with trypsin, papain, andproteinase K (5:1 ratio SmtA/protease). Digestion patterns of MtsAwith/without pretreatment with EDTA were compared, to elucidate whethera potentially present cation ligand affected the conformation of theprotein so as to change accessibility of proteolytic cleavage sites. Nosuch effect was seen (data not shown). A relative resistance toproteolytic digestion with papain was noted, in accordance with the caseof the native protein (see above). In addition a high degree of trypsinresistance was noted.

We chose to investigate direct binding of metal radioisotopes (⁵⁹Fe,⁶⁵Zn and 54Mn) to the recombinant protein. Iron proved to be notoriouslydifficult to work with, since ferric iron is essentially insoluble atneutral pH. Precipitation problems therefore prevented reasonableinterpretation of several assays tried. Copper radioisotopes decay toorapidly to handle practically. Interestingly, an interaction with Zncould be demonstrated. GST and MtsA were incubated with ⁶⁵Zn, in thepresence of 0.25 M NaCl, and then subjected to gel filtration. Fractionswere collected, and their protein concentration and radioactivity wasmeasured. The result (FIG. 3A) indicates comigration of ⁶⁵Zn with MtsA,but not with GST. When the molar amount of comigrating ⁶⁵Zn is comparedwith the protein content, a 60% saturation of the protein is found(assuming a single binding site). Similarly incubated GST and MtsA weresubjected to native PAGE, and then autoradiography. Radioactivity wasseen at the place of MtsA, but not for GST (data not shown), and thiscould be inhibited by adding ZnCl₂ to the initial incubation.

A slot blot assay was also performed, applying MtsA and GST onto anitrocellulose membrane in dilution series. The membrane was thenincubated with ⁶⁵Zn, and, following washing steps, radioactivity wasfound in the MtsA fusion sample, but not in the GST sample (data notshown). This interaction could be efficiently inhibited (88%) byaddition of 1 mM ZnCl₂ during incubation. A varying degree of inhibitionwas seen when adding other metal salts. Among these, only Cu(II)inhibited the interaction to a high degree (74%). A similar experimentwas performed with ⁵⁹Fe(III), which also bound to MtsA but not to GST(data not shown). The binding was completely inhibited by addition of 1mM Fe(III) citrate, but only weakly inhibited by 1 mM CuCl, and MnCl₂.Addition of Fe(II)-sulfate or ZnCl₂ caused unspecific interaction withthe membrane, probably due to precipitation. All these assays were alsoperformed with ⁵⁴Mn, but no interaction between the radioisotope and anyof the proteins was found.

Example 6 Gene and Protein Prevalence

Genomic DNA was isolated and subjected to PCR, using the primers aslisted, and an annealling temperature of 45 degrees. The primers usedfor PCR amplification of MtsA gene included: LUND MTSA (SEQ ID No 3)5′-TAG-TAG-CGA-ATT-CGT-CGA-CTG-GCG-CTA-3′ MTSA 4 (SEQ ID No 4)5′-AGC-ACA-ACT-CGA-GAA-TCG-CTG-TGC-TTT-A-3′

The PCR reagents and reagent volumes used for each sample were asfollows: 10 μl GeneAmp 10×PCR Buffer II & MgCl₂, 5 μl LUND MTSA primer10 μM, 5 μl MTSA 4 primer 10 μM, 10 μl DNTP mixture 2.5 mM (dATP, dCTP,dGTP, dTTP), 67.5 μl dH₂O, 2 μl Bacterial DNA template, 0.5 μl AmpliTaqGold DNA polymerase. The thermocycler PCR settings were as follows: A.94° C. for 10 minutes, B. 95° C. for 1 minute, C. 45° C. for 1 minute,D. 74° C. for 1 minute, E. 74° C. for 7 minutes, F. 0° C. tubes storedindefinitely. Steps B-D were cycled 35 times. All 23 strains havedetectable PCR product in the predicted size range. The results are setout in Table 1 below.

Sequence Conservation

The MtsA PCR products from three strains of GAS have been sequenced,resulting in approximately 750 bp from each. The sequences showed a highlevel of conservation, compared to the sequence of the original AP1strain. The strains sequenced were M1 API, T11 AP74 and M1 3686-98. Thesequencing primers were identical to those used for the PCR reaction.

Example 7 Generation of Antiserum

From the genbank MtsA sequence, a number of peptides have been selectedbased on predicted antigenicity. These are as follows: Name Sequence(including terminal C) length Spy-LP-TDS21 C*TDSIAKKGKP GDSYYAMMKWN-COOH22 (259-280) Spy-LP-ESS16 C*ESSVDRRPME TVSKDS-COOH 17 (234-250)Spy-LP-KQL17 C*KQLIAKDPKN KETYEKN-COOH 18 (136-153) Spy-LP-EIN19C*EINTEEEGTP DQISSLIEK-COOH 20 (204-223)

A further peptide, QDP13, has also been highlighted as a potentialvaccine candidate, based on it's ability to raise antisera in rabbitswhich reacts with the MtsA protein (data not shown). QDP13 QDPHEYEPLPEDV13

The first four peptides have been conjuguated to KLH and used toimmunize two sheep each as follows: the peptides were derived frompeptide synthesis by Genosys Biotechnologies Inc. They are supplied asconjugated Iyophilized powder in 0.5 mg aliquots in capped vials. Thepeptides were conjugated to keyhole limpet hemocyanin. C* is a cysteineinsert for attachment to a hetero bifunctional linker.

In addition, there were BSA-conjugates of the peptides. They were storedas lyophilized powder in 0.5 mg aliquots in capped vials. TheBSA-conjugates were used for ELISA plate immobilization/evaluation.

For each immunisation the following regent mixture was administered:

-   -   A fixed volume of 3.25 ml Freund's complete/incomplete adjuvant.    -   1 mg of peptide conjugate in 1.3 ml volume of saline for the        primary immunisation at day 0 or 0.5 mg of peptide conjugate in        1.3 ml volume of saline for the subsequent immunisations. The        immunisation mixture was achieved by emulsifying the        peptide-conjugates in saline with the Freund's adjuvant. The        immunisation mixture was injected into 6 subcutaneous sites for        each sheep.

These antisera have been tested by ELISA for their ability to bind tothe immunizing peptides. Peptide was coated onto microtitre plates (100μl/well) at a concentration of 5 μg/ml, in 0.05M carbonate-bicarbonatebuffer pH 9.6. The plates were incubated for 1 hour at 37° C. The plateswere then washed×5 with PBS-T (250 μl/well) and blocked with 1%BSA/PBS-T (100 μl/well) for 1 hour at 37° C.

After washing the plates×3 with PBS-T, pre and post immune sera fromsheep immunised with peptide conjugate vaccine candidates includingFCA/Spy-LP-TDS21-KLH, FCA/Spy-LP-ESS16-KLH, FCA/Spy-LP-KQL17-KLH andFCA/Spy-LP-EIN19-KLH were diluted 1/10,000 with PBS-T. The sera werethen incubated on plates coated with the corresponding peptide (100 μlsera/well) for 1 hour, at 37° C. The plates were washed×3 with PBS-T andincubated with donkey anti-sheep IgG/peroxidase conjugate (1/1000 inPBS-T) for 1 hour at 37° C. After washing×5 with PBS-T, the plates wereincubated with 0.1 mg/ml TMB substrate (100 μl/well) for 10 minutes andthen the reaction was stopped with 2M H₂SO₄ (50 μl/well). Absorbanceswere read at 450 nm.

FIG. 4 shows that both sheep immunized with each peptide raised strongantisera against the corresponding peptide (eight sheep in total). FIG.5 shows that the anti-peptide antisera also bound to whole MtsA protein,demonstrating the ability of the peptides to raise antisera which maybind to natural protein on cells. That this binding is specific wasdemonstrated in a peptide blocking ELISA experiment, where peptide wastitrated into the sera prior to reacting the sera in an ELISA againstwhole protein (FIG. 6).

Example 8 Western Blot Analysis

Various strains of S.pyogenes were tested by Western Blot, using thesheep anti-TDS 21 antisera to detect the protein. In particular, WesternBlots from SDS-PAGE of S.pyogenes proteins were incubated with 30 ml 5%(W/V) milk powder/PBS as a blocking solution. 30 μl (1/1000) sheepantiserum (anti-TDS21) was added in 30 ml 5% (W/V) milk powder PBS andincubated for 60 mins at room temperature. After 3 washes, 3.0 μl(1/10000) of donkey anti-sheep IgG peroxidase in 30 μl 5% (W/V) milkpowder/PBS was added and incubated for 60 minutes, washed and peroxidasesubstrate added. All 23 strains had detectable protein.

The results along with the PCR results are set out below. TABLE 1 MTSAGene and Protein Distribution in group A Streptococcus, as determined byPCR analysis and Western Blot. Protein MtsA Approximate Predicted molexpression Group A gene No. of base weight of (Western Strain REF. NoSource detection pairs protein Blot) M1 AP-1 LUND + 949.05 31.64 + T11AP-74 LUND + 949.05 31.64 + M1 3686-98 CDC + 993.51 33.12 + M2 3688-98CDC + TBD TBD + M3 3671-98 CDC + 943.87 31.46 + M4 3670-98 CDC + 943.8731.46 + M5 3667-98 CDC + 890.90 29.69 + M6 3693-98 CDC + TBD TBD + M93261-98 CDC + 890.90 29.69 + M11 3904-98 CDC + 890.90 29.69 + M123664-98 CDC + 890.90 29.69 + M18 3258-98 CDC + TBD TBD + M19 3913-98CDC + 840.90 28.03 + M22 3858-98 CDC + 840.90 28.03 + M24  55-987 CDC +TBD TBD + M28 3272-98 CDC + 840.90 28.03 + M49 3274-98 CDC + 840.9028.03 + M55  189-98 CDC + TBD TBD + M57  55-790 CDC + 840.90 28.03 + M60 978-97 CDC + 793.70 26.45 + M non  88/30 Australia + 840.90 28.03 +typeable M6 2036 Australia + TBD TBD + M12 2040 Australia + TBD TBD ++ means genomic DNA PCR product or protein was detectable,TBD = to be determined.

Example 9 Immunisation Studies

The Spy-LP-ESS16 peptide was also used to immunize rats. The rats weretreated intraperitoneally with a dose volume of 1 ml/animal. Controlgroups received 1 ml saline, 100 μg keyhole limpet hemocyanin (KLH) in 1ml saline, 500 μl of Freund's complete adjuvant (FCA) plus 500 μl salineor 1 mg aluminum hydroxide in 1 ml total volume. Peptide wasadministered at 100 μg/ml. Rats were given a boost on day 21 and bled onday 42. Plates were coated with peptide at 5 μg/ml in carbonate buffer.Serum was titrated against the peptide in 10 fold dilutions from 1 in100 in PBS and binding detected using an anti-rat HRP conjugate and TMBsubstrate. To summarize this data, the control sera, sera from ratsinjected with free peptide in the absence of adjuvant, sera from ratsinjected with free peptide in FCA, and sera from rats injected with freepeptide in alhydrogel (alum) did not react to the ESS16 peptide bound toplates. In contrast, sera from rats injected with peptide KLH conjugatesor peptide KLH conjugates in alum or CFA all contained substantialantibody titres.

Example 10 Analysis of Natural Human Immunity

The TDS21 peptide was chosen for further studies of endemic humanimmunity. Free TDS21 peptide was coated onto ELISA plates and reactedwith sera obtained from Thai volunteers. The Thai population under studyhas a high prevalence of infection with GAS, and a high incidence ofrheumatic heart disease (RHD). Normal and RHD responses were compared.The results showed clearly that both normal and RHD sera may containantibodies to TDS21, and in fact the incidence of such antibodiesappears to be higher in the normal population (data not shown). Thus,the TDS21 epitope is recognized during natural exposure to GAS.

Similarly, the TDS21 and QDP13 peptides were used to stimulatelymphocytes from the same Thai individuals. As with antibodies, theTDS21 response was more common in the control than the RHD population.QDP13 stimulated only cells from RHD individuals.

1-3. (canceled)
 4. A polynucleotide having a sequence which is (i) thenucleotide coding sequence of SEQ ID NO 1 or the sequence complementarythereto, (ii) a nucleotide sequence which selectively hybridises to asaid sequence (i) or a fragment thereof, or (iii) a nucleotide sequencewhich codes for a polypeptide having the same amino acid sequence asthat encoded by a said sequence (i) or (ii).
 5. A polynucleotideaccording to claim 4 wherein the sequence (i), (ii) or (iii) encodes apolypeptide capable of generating an immune response to a Streptococcus.6. An expression vector comprising a polynucleotide according to claim 4operably linked to a regulatory sequence.
 7. A host cell transformedwith a polynucleotide of claim
 4. 8. A process of producing apolypeptide suitable for use in vaccination against a Streptococcuscomprising maintaining the host cell as defined in claim 7 underconditions to provide expression of the polypeptide and recovering theexpressed polypeptide.
 9. An antibody specific for a polypeptide whichcomprises: (a) the amino acid sequence of SEQ ID NO 2, (b) a variant of(a) which is capable of binding an anti-MtsA antibody or (c) a fragmentof (a) or (b) of at least 6 amino acids in length which is capable ofbinding an anti-MtsA antibody. 10-12. (canceled)